Pursuant to 37 C.F.R. xc2xa7 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Not Applicable.
The present invention relates to fluid dispensing systems. More specifically, the invention provides automated systems for simultaneously producing multiple fluid mixtures in multiple multiwell plates with high throughput.
Acquiring knowledge of the detailed three-dimensional structures of proteins and other macromolecules is central to structure-based drug development. A prominent methodology for solving high-resolution molecular structure is x-ray crystallography, which entails crystallizing the molecule under consideration. The process typically involves crystallizing a test sample that includes the target molecule in a fluid mixture formulated to provide stable and highly ordered crystals. The art of crystallization, however, is often difficult and time consuming. For example, each new protein crystallization generally requires a unique concentration and mixture of salts, precipitants, and other fluids for crystal growth to occur. It is typically necessary to screen a protein sample against hundreds or even thousands of varied fluid mixtures or crystallization mother liquors in order to identify the proper combination of fluids that will yield a crystallized form of the protein. To further illustrate, finding the proper fluid mixture may require varying the composition of the mixture using a multi-dimensional array of variables, such as different types of aqueous, salt, precipitant, organic, and buffer solutions, different concentrations and pH levels for those fluids, different atmospheric conditions, and the like.
Screens for suitable crystallization conditions are currently conducted manually using skilled technicians. Performing each screen is generally a labor intensive process, in part, because the different fluid mixtures into which the target molecules are deposited must themselves be dispensed, e.g., in very small amounts into the wells of multiwell plates, such as microwell plates. The physical act of dispensing these small amounts into such small fluid containers is itself a time consuming and inaccurate process. In addition, the amount of test sample available for each individual screen is often limited. Further, the screening fluids used in each screen are typically measured in microliter volumes or less. This requires a high level of precision and accuracy that can be difficult even for skilled technicians. The reliability and reproducibility of each screen are integral to the precision and accuracy of the screens. Accordingly, there exists a need to automate the screening process to increase throughput, and to increase the level of precision, accuracy, and reproducibility of the process.
As mentioned, conventional crystallization techniques generally require that each test sample to be crystallized be screened against numerous different fluid mixtures in order to find a proper composition that provides stable crystallization conditions for the particular target molecule in question. In a manual screening process, a technician is primarily responsible for measuring, mixing, and dispensing each unique fluid mixture. Such a manual process is time consuming and expensive, and therefore the variations of fluid mixtures are often limited because of time constraints in the screening process. Unfortunately, by reducing the granularity of the screen, a less than optimum fluid mixture will likely be selected. Further, such a manual screening process is highly susceptible to human mathematical and measurement errors in fluid preparation. As a consequence, the screen may yield erroneous, unreliable, and non-reproducible results.
Yet another problem associated with screening crystallization conditions is that many of the component fluids of crystallization mother liquors used in the screens are highly volatile. These volatile fluids can evaporate or change in character rapidly in a short period of time. Therefore, it is often difficult to manually prepare a screen that includes a large number of individual crystallization assays due to the time required to deposit the fluids into each well. As the different fluids are deposited in each well, the volatile fluids can evaporate or otherwise change composition, rendering the particular screen inaccurate or otherwise biased.
From the above, it is apparent that there is a substantial need for fluid dispensing systems that simultaneously produce multiple fluid mixtures (e.g., mother liquor solutions for crystallization screens, etc.) in multiple multiwell plates. These and a variety of additional features of the present invention will become evident upon complete review of the following disclosure.
The present invention provides highly automated fluid delivery systems and related methods with significantly improved throughput relative to preexisting technologies. In particular, the invention relates to a fluid dispensing system that simultaneously produces multiple fluid mixtures on-the-fly in multiple multiwell plates. The invention also includes software that, inter alia, directs fluid dispensing from multiple fluid dispensers and tracks fluid mixture compositions in the wells of multiwell plates. In addition, various systems and related methods are also provided for performing assorted downstream processes. Fluid mixtures prepared utilizing the systems and methods described herein are useful for essentially any purpose including, for example, preparation of mother liquor solutions for high throughput crystallization screens.
The present invention provides a fluid dispensing system that includes an array of fluid dispensers. In some embodiments, the array includes at least two fluid dispensers that are spaced at least a sufficient distance apart to simultaneously dispense a fluid into a well of a first multiwell plate and a corresponding well of a second multiwell plate when both plates are placed beneath the array of fluid dispensers. The number of fluid dispensers in the array is, in certain embodiments, at least as great as the number of wells in two lines of wells of a single multiwell plate; and in these embodiments the fluid dispensers are spaced an appropriate distance apart from one another to simultaneously dispense a fluid into wells of multiple multiwell plates when the plates are placed underneath the fluid dispensers. In some configurations, the array of fluid dispensers includes a plurality of linear arrays, each of which comprises at least two fluid dispensers that are spaced at least a sufficient distance apart to simultaneously dispense a fluid into a well of a first multiwell plate and a corresponding well of a second multiwell plate when both plates are placed beneath the array of fluid dispensers.
In one aspect, the present invention provides a fluid dispensing system that includes a linear array of fluid dispensers in which the linear array includes a number of fluid dispensers that is greater than the number of wells in a line of wells (e.g., a row or column of wells) of a single multiwell plate, which line of wells is parallel to a longitudinal axis of the linear array. Each dispenser is spaced an appropriate distance apart from an adjacent fluid dispenser to allow the two adjacent dispensers to simultaneously dispense a fluid into adjacent wells of a multiwell plate when the multiwell plate is placed underneath the fluid dispensers. Suitable spacings include, but are not limited to, 144 mm, 72 mm, 36 mm, 18 mm, 9 mm, 4.5 mm, 2.25 mm or less (center to center), depending upon the plate format. In some embodiments, the number of fluid dispensers is x times the number of wells in the line of wells of a single multiwell plate and x is a whole number greater than or equal to 2 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more), although other configurations can be utilized. Typically, each of the fluid dispensers is connected to a fluid container that contains a different fluid (e.g., a different stock solution, such as water, a precipitant or polymer solution, a buffer, an organic solution, or the like) for producing varied mixtures of crystallization mother liquor solutions. Furthermore, although other volumes are optionally dispensed, each fluid dispenser separately generally dispenses selected volumes between about 1 nl and about 500 xcexcl. The linear array of fluid dispensers is optionally configured so that the fluid dispensers can deliver fluid to at least two, at least three, at least four, or more multiwell plates at the same time.
To illustrate, the adjacent dispensers are optionally spaced an appropriate distance apart to allow the adjacent fluid dispensers to simultaneously dispense a fluid into adjacent wells of a 96-well plate. For example, if the line of wells that is parallel to a longitudinal axis of the linear array includes eight wells, then the linear array comprises at least nine fluid dispensers. In contrast, if multiwell plate is oriented such that the line of wells that is parallel to the longitudinal axis of the linear array includes twelve wells, then the linear array comprises at least thirteen fluid dispensers. For example, the linear array optionally includes various numbers of fluid dispensers (e.g., at least 17 fluid dispensers, at least 25 fluid dispensers, at least 33 fluid dispensers, etc.). In some embodiments, the linear array includes 96 fluid dispensers.
In some embodiments, the fluid dispensing system includes an array of fluid dispensers comprising more than 12 fluid dispensers aligned with each other along a longitudinal axis such that two adjacent fluid dispensers can each dispense a fluid into a different adjacent well of a multiwell plate positioned stationary underneath the array of fluid dispensers; and a mechanism for moving the array of fluid dispensers and a multiwell plate positioned underneath the array of fluid dispensers relative to each other in a direction perpendicular to the longitudinal axis. The fluid dispensing systems can include, for example, 16, 24, 48, 60, 96, or more fluid dispensers aligned with each other along a longitudinal axis. The fluid dispensers in the fluid dispensing system are, in some embodiments, in fluid connection with at least 8 different fluid sources. For example, the fluid dispensers can be in fluid connection with 8, 16, 24, 48, 60, 96 or more different fluid sources. The fluid dispensers can, in some embodiments of the fluid dispensing system, deliver fluid to wells of at least three different multi-well plates at the same time. For example, the invention provides fluid dispensing systems in which the fluid dispensers can deliver fluid to wells of 3, 4, 5, 6, or more different multi-well plates at the same time.
In certain embodiments, at least a first fluid dispenser in the array is connected to a fluid container that contains a first fluid and at least a second fluid dispenser in the array is connected to a fluid container that contains a second fluid that differs from the first fluid. Adjacent fluid dispensers typically dispense different fluids. For example, the first and second fluids are independently selected from, e.g., water, a stock solution, a buffer, a reagent, a solvent, a salt solution, a polymer solution, an inorganic solution, an organic solvent, a cell suspension, or the like. In some embodiments, at least a first fluid dispenser in the array is connected to a fluid container that contains water, at least a second fluid dispenser in the array is connected to a fluid container that contains a salt solution, at least a third fluid dispenser in the array is connected to a fluid container that contains a polymer solution, and at least a fourth fluid dispenser in the array is connected to a fluid container that contains an organic solvent. The salt solution optionally includes one or more components selected from, e.g., cacodylic acid, CHES, HEPES, citric acid, malonic acid, MES, phosphoric acid, acetic acid, a salt thereof, or the like. The polymer solution optionally includes one or more components selected from, e.g., glycerol, ethylene glycol, formate, spermine, polyethylene glycol, or the like. The organic solvent optionally includes one or more components selected from, e.g., 1,2-propanediol, DMSO, methanol, dioxane, trifluoroethanol, MPD, ethanol, isopropanol, or the like. In certain embodiments, each of these salt solutions, polymer solutions, and organic solvents is contained in at least one fluid container which is connected to a fluid dispenser in the array.
At least one, but typically each, of the fluid dispensers includes (i) a fluid conduit (e.g., a flexible tube or the like) in fluid communication with a fluid source or reservoir, and (ii) a pump (e.g., a peristaltic pump, a syringe pump, etc.) operably connected to the fluid conduit to convey fluid through the fluid conduit from the fluid source to the wells of the multiwell plates. In addition, each of the fluid dispensers generally includes a solenoid valve or a piezoelectric valve that operates in coordination with the pump to dispense selected volumes of fluid.
In preferred embodiments, a fluid dispensing system of the invention also includes a moving element (e.g., a conveyor belt, etc.) that moves the multiwell plates in a direction parallel to a longitudinal axis of the array of the fluid dispensers (e.g., in an x-axis direction). Optionally, the moving element moves reversibly (e.g., either in a positive or negative x-axis direction) at a given time. The moving element typically has a length of at least n of the multiwell plates, wherein n is the number of the multiwell plates. In certain embodiments, for example, n is a whole number selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In addition, the array of fluid dispensers typically includes a support structure that is operably connected to a drive mechanism (e.g., a stepper motor, a servo motor, or the like) that reversibly moves the support structure in a direction perpendicular to a direction of movement of the moving element (e.g., in a positive or a negative y-axis direction at a given time). Optionally, fluid dispensing systems further include one or more cleaning devices to clean the fluid dispensers. Fluid dispensing systems also optionally include one or more waste containers into which volumes of waste fluid are dispensed from the dispensers.
A fluid dispensing system of the invention generally further includes a controller that is operably connected to the fluid dispensers, the moving element, and the drive mechanism, which controller controls at least fluid dispensation from the fluid dispensers, moving element movement, and support structure movement. The controller typically includes a logic device and a database. The logic device generally includes control system software and machine software in which the control system software communicates with the machine software and the database to control execution of the machine software. In addition, the machine software typically includes one or more logic instructions that direct the fluid dispensing system to, e.g., move the moving element a selected distance, move the support structure a selected distance, and dispense selected volumes of fluids from selected fluid dispensers into selected wells of the multiwell plates. Optionally, the logic instructions direct the fluid dispensing system to (a) sequentially position each well of two or more multiwell plates underneath each of the fluid dispensers, and (b) dispense selected volumes of fluid from selected fluid dispensers into selected wells of the multiwell plates when the selected fluid dispensers are positioned above the selected wells, thereby simultaneously producing multiple fluid mixtures in two or more multiwell plates. The system control logic can direct when fluid is dispensed from a given fluid dispenser to a well of a multiwell plate based on a position of the multiwell plate in the system. For some or all wells in a particular row, the selected volume dispensed by a selected fluid dispenser can be zero if the fluid mixture being prepared in that well is not intended to contain the fluid being dispensed by that fluid dispenser.
As an additional option, the logic instructions direct the fluid dispensing system to position a first well of a first row of a multiwell plate under a first fluid dispenser, move the support structure sequentially across the entire first row of a multi-well plate, dispensing selected volumes of a first fluid from the first fluid dispenser into one or more selected wells in the first row when the first fluid dispenser is positioned above the selected well, sequentially advance the moving element to position a first well of a second row of a multiwell plate under the first fluid dispenser and the first well of the first row under a second fluid dispenser, and move the support structure sequentially across the entire first and second rows, dispensing selected volumes of a first fluid from the first fluid dispenser into one or more selected wells in the second row when the first fluid dispenser is positioned above the selected well, and dispensing selected volumes of a second fluid from the second fluid dispenser into one or more selected wells in the first row when the second fluid dispenser is positioned above the selected well. The database typically includes information about, e.g., fluids in fluid containers that are in fluid communication with the fluid dispensers, selected wells into which a selected fluid dispenser is to dispense a selected fluid in which the selected wells are located on two or more multiwell plates, and selected volumes of the selected fluids that are to be dispensed into each selected well.
In another aspect, the present invention relates to methods of simultaneously producing multiple fluid mixtures in multiple multiwell plates. The methods include (a) providing a fluid dispensing system that includes an array of fluid dispensers, wherein the array comprises at least two fluid dispensers that are spaced at least a sufficient distance apart to simultaneously dispense a fluid into a well of a first multiwell plate and a corresponding well of a second multiwell plate when both plates are placed beneath the array of fluid dispensers. In some embodiments, the method uses a fluid dispensing system that includes a linear array of fluid dispensers in which each dispenser is spaced an appropriate distance apart from an adjacent dispenser to allow the two adjacent dispensers to simultaneously dispense a fluid into adjacent wells of a multiwell plate, and the number of fluid dispensers in the linear array is greater than the number of wells in a line of wells (e.g., a row or column of wells) of the multiwell plate that is parallel to a longitudinal axis of the linear array. The number of fluid dispensers, in some embodiments, is x times the number of wells in the line of wells of the multiwell plate and x is a whole number greater than or equal to 2 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more), although this relationship between number of wells and number of fluid dispensers is not required. The methods also include (b) sequentially positioning each well of the multiple multi well plates underneath each of the fluid dispensers. In addition, the methods include (c) dispensing selected volumes (e.g., between about 1 nl and about 500 xcexcl) of fluid from selected fluid dispensers into selected wells of the multiple multiwell plates when the selected fluid dispensers and the selected wells are positioned above the selected wells to simultaneously produce the multiple fluid mixtures in the multiple multiwell plates. In preferred embodiments, each of the fluid dispensers dispenses a different fluid. Generally, one or more of the fluid mixtures include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mixed fluids. In certain embodiments, the methods further include cleaning the fluid dispensers after one or more of the selected volumes are dispensed and/or dispensing volumes of waste fluid from the fluid dispensers into waste containers that correspond to linearly arrayed portions of the fluid dispensers.
In another aspect, the invention features a system for efficiently preparing mother liquors in a plurality of multi-well sample plates for, e.g., a coarse screen or a fine screen, in a plurality of sample plates. The plurality of sample plates is arranged with corresponding columns aligned, the system including: (a) a plate arranging area configured to receive the plurality of sample plates; (b) a plurality of fluid containers, each fluid container holding a stock solutions; (c) a plurality of fluid dispensers arranged in an array, each fluid dispenser being in fluid communication with an associated one of the fluid containers; (d) a drive mechanism constructed to sequentially position the fluid dispensers in the array directly over each column of wells in the sample plate; (e) a dispensing mechanism associated with each fluid dispenser; and (f) a fluid controller communicating to the dispensing mechanism in which the fluid controller directs selected dispensing mechanisms to deliver a quantity of each associated mother liquor into each selected sample well in a column before the drive mechanism moves the fluid dispenser array to a next column.
In preferred embodiments the plurality of fluid dispensers are configured so that 2, 3, 4, 5, 6, 7 or 8 sample plates can be beneath the plurality of fluid dispensers at the same time. The plurality of fluid dispensers preferably are configured so that the fluid dispensers can deliver the material to 2, 3, 4, 5, 6, 7 or 8 sample plates at the same time. The plurality of fluid dispensers may be configured so that all of the fluid dispensers can deliver the material at the same time. In one preferred embodiment, the system includes a moving element that has a length of at least n sample plates, wherein n is the number of sample plates, wherein each sample plate has m wells, wherein m is the number of wells, wherein the system processes a sample plate every m dispensings even though the sample plate is in the system for n times m dispensings. For example, the moving element has a length of at least five sample plates, wherein each sample plate has 96 wells, wherein the system processes a sample plate every 96 dispensings, even though the sample plate is in the system for 480 dispensings. The fluid controller preferably directs the delivery of the material from each fluid container to each sample plate, for example, the dispenser controller directs the delivery of the material from each of at least eight fluid containers to each of at least five multi-well plates.
In another aspect, the present invention provides a method for automatically preparing a mixture in a well of a multi-well holder. The method involves the steps of: (a) moving the multi-well holder so that the well is positioned below a fluid dispensing device; (b) dispensing fluid from the fluid dispensing device into the well; and (c) repeatedly moving the multi-well holder so that the well is positioned below a next fluid dispensing device and dispensing fluid from the next fluid dispensing device into the well until a predetermined mixture is prepared.
In preferred embodiments, the plurality of fluid dispensers are configured so that 2, 3, 4, 5, 6, 7 or 8 multi-well holders can be beneath the plurality of fluid dispensers at the same time. The plurality of fluid dispensers preferably are configured so that the fluid dispensers can deliver the material to 1, 2, 3, 4, 5, 6, 7 or 8 multi-well holders at the same time. The plurality of fluid dispensers may be configured so that all of the fluid dispensers can deliver the material at the same time. In one preferred embodiment, the sample plates are on a moving element that has a length of at least n sample plates, wherein n is the number of multi-well plates, wherein each multi-well plate has m wells, wherein m is the number of wells, wherein the method processes a multi-well plate every m dispensings even though the method involves n times m dispensings. For example, the sample plates are on a moving element that has a length of at least five multi-well plates, wherein each multi-well plate has 96 wells, wherein the method processes a multi-well plate every 96 dispensings, even though the method involves 480 dispensings. Preferably, a controller directs the delivery of the material from one or more fluid containers to each sample plate. For example, the controller directs the delivery of the material from each of at least eight fluid containers to each of at least five multi-well plates.
Finally, in another aspect, the present invention provides a fluid dispenser array for dispensing liquid into a plurality of multi-well sample plates. The fluid dispenser array includes a plurality of N fluid dispensers coupled into a linear array. N is a whole number multiple of the number of sample wells in one line of each sample plate. Each sample plate includes sample wells organized in a geometric pattern. The line may be a row or a column. By way of example, the number of sample wells in a line may be 12 and N may be 96.
In preferred embodiments of any of the aspects of the invention described herein, the footprint of the tubes in the column direction (i.e., the column length footprint) of the multi-well holder is at least 5.030, 10.060, 15.090, 20.120, 25.150, 30.180, 35.210, 40.240, or 45.270 inches long.
It readily will be appreciated that an advantage of the present system is to increase the speed, accuracy and reliability of protein crystallization and processing operations.